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diversification of nuclear receptors
Brice Beinsteiner, Gabriel Markov, Stéphane Erb, Yassmine Chebaro, Alastair
Mcewen, Sarah Cianférani, Vincent Laudet, Dino Moras, Isabelle Billas
To cite this version:
origin and diversification of nuclear receptors
Brice BeinsteinerID1,2,3,4‡, Gabriel V. MarkovID5‡, Ste´phane ErbID6, Yassmine ChebaroID1,2,3,4, Alastair G. McEwenID1,2,3,4, Sarah Cianfe´rani6, Vincent LaudetID7*, Dino MorasID1,2,3,4*, Isabelle M. L. BillasID1,2,3,4*
1 IGBMC (Institute of Genetics and of Molecular and Cellular Biology), Illkirch, France, 2 Universite´ de Strasbourg, Unistra, Strasbourg, France, 3 Institut National de la Sante´ et de la Recherche Me´dicale (INSERM) U1258, Illkirch, France, 4 Centre National de la Recherche Scientifique (CNRS) UMR 7104, Illkirch, France, 5 Sorbonne Universite´, CNRS, UMR 8227, Integrative Biology of Marine Models, (LBI2M, UMR8227), Station Biologique de Roscoff (SBR), Roscoff, France, 6 Laboratoire de Spectrome´trie de Masse BioOrganique, Universite´ de Strasbourg, CNRS, IPHC UMR 7178, Strasbourg, France, 7 Marine Eco-Evo-Devo Unit. Okinawa Institute of Science and Technology, Onna-son, Okinawa, Japan
‡ co-first authors
*vincent.laudet@oist.jp(VL);moras@igbmc.fr(DM);billas@igbmc.fr(IMLB)
Abstract
Nuclear receptors are ligand-activated transcription factors that modulate gene regulatory networks from embryonic development to adult physiology and thus represent major targets for clinical interventions in many diseases. Most nuclear receptors function either as homo-dimers or as heterohomo-dimers. The dimerization is crucial for gene regulation by nuclear recep-tors, by extending the repertoire of binding sites in the promoters or the enhancers of target genes via combinatorial interactions. Here, we focused our attention on an unusual struc-tural variation of theα-helix, calledπ-turn that is present in helix H7 of the ligand-binding domain of RXR and HNF4. By tracing back the complex evolutionary history of theπ-turn, we demonstrate that it was present ancestrally and then independently lost in several nuclear receptor lineages. Importantly, the evolutionary history of theπ-turn motif is parallel to the evolutionary diversification of the nuclear receptor dimerization ability from ancestral homodimers to derived heterodimers. We then carried out structural and biophysical analy-ses, in particular through point mutation studies of key RXR signature residues and showed that this motif plays a critical role in the network of interactions stabilizing homodimers. We further showed that theπ-turn was instrumental in allowing a flexible heterodimeric interface of RXR in order to accommodate multiple interfaces with numerous partners and critical for the emergence of high affinity receptors. Altogether, our work allows to identify a functional role for theπ-turn in oligomerization of nuclear receptors and reveals how this motif is linked to the emergence of a critical biological function. We conclude that theπ-turn can be viewed as a structural exaptation that has contributed to enlarging the functional repertoire of nuclear receptors. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS
Citation: Beinsteiner B, Markov GV, Erb S, Chebaro
Y, McEwen AG, Cianfe´rani S, et al. (2021) A structural signature motif enlightens the origin and diversification of nuclear receptors. PLoS Genet 17(4): e1009492.https://doi.org/10.1371/journal. pgen.1009492
Editor: Artyom Kopp, University of California Davis,
UNITED STATES
Received: December 14, 2020 Accepted: March 15, 2021 Published: April 21, 2021
Peer Review History: PLOS recognizes the
benefits of transparency in the peer review process; therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. The editorial history of this article is available here:
https://doi.org/10.1371/journal.pgen.1009492 Copyright:© 2021 Beinsteiner et al. This is an open access article distributed under the terms of the
Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability Statement: The data and R script
Author summary
The origin of novelties is a central topic in evolutionary biology. A fundamental question is how organisms constrained by natural selection can divert from existing schemes to set up novel structures or pathways. Among the most important strategies are exaptations, which represent pre-adaptation strategies. Many examples exist in biology, at both mor-phological and molecular levels, such as the one reported here that focuses on an unusual structural feature called theπ-turn. It is found in the structure of the most ancestral nuclear receptors RXR and HNF4. The analyses trace back the complex evolutionary his-tory of theπ-turn to more than 500 million years ago, before the Cambrian explosion and show that this feature was essential for the heterodimerization capacity of RXR. Nuclear receptor lineages that emerged later in evolution lost theπ-turn. We demonstrate here that this loss in nuclear receptors that heterodimerize with RXR was critical for the emer-gence of high affinity receptors, such as the vitamin D and the thyroid hormone receptors. On the other hand, the conservedπ-turn in RXR allowed it to accommodate multiple het-erodimer interfaces with numerous partners. This structural exaptation allowed for the remarkable diversification of nuclear receptors.
Introduction
The nuclear hormone receptor (NR) superfamily includes receptors for hydrophobic ligands such as steroid hormones, retinoic acids, thyroid hormones or fatty acids derivatives [1,2]. This superfamily, which clusters 48 genes in human, is subjected to an intense scrutiny because of the essential role played by NRs in animal development, metabolism and physiology. NRs are important drug targets since dysfunctions of homeostasis and signaling pathways con-trolled by these receptors are associated with many diseases including cancer, metabolic syn-drome or reproductive failure [3].
All nuclear receptor proteins share a characteristic modular structure that consists of con-served DNA and ligand binding domains (DBD and LBD, respectively) separated and flanked by poorly conserved flexible regions [1,2]. Typically, distant NRs exhibit ca. 60% sequence identity in their DBD and 30% in their LBD. The availability of the ligand controls NR activity in space and in time since ligand binding inside a specific pocket within the LBD induces a conformational change of the receptor allowing the release of corepressors, the recruitment of coactivators and the transactivation of target genes [1,2].
Given their importance and also because their long conserved domains are favorable for phylogenetic analysis, the origin of the NR superfamily have been scrutinized for a long time, allowing to define distinct subfamilies [4–6]. Full NRs are specific to animals whereas DBD sequences have been found in the genomes of some choanoflagellates, the closest metazoan rel-atives [7]. After several lineage-specific events of gene loss or gene duplications, the size of the superfamily ranges from about 20 members in insects to about 48 to 70 in vertebrates, with a specific expansion in some lineages such as nematodes for which more than 260 NR genes are present [8–10].
The analysis of complete genome sequences available in a number of animal species, includ-ing early metazoans such as sponges, placozoans or cnidarians have allowed a better under-standing of the first step of NR diversification. The observation that sponges, which despite some controversy are believed to be the earliest metazoan phyla [11] contains only two NR genes, called here SpNR1 and SpNR2, have shed a decisive light on the first steps of NR evolu-tion [9]. This has allowed the positioning of the root of the NR tree within subfamily II that in within the manuscript and itsSupporting
Informationfiles.
Funding: This work was supported by the Agence
Nationale de la Recherche [Grant Number ANR-2010-BLAN-1234 01] (V.L., I.M.L.B, D.M.), by the Universite´ de Strasbourg (Unistra) (I.M.L.B., D.M., S.E., A.G.M, B.B, Y.C.), CNRS (I.M.L.B., D.M., S.C., A.G.M, B.B, Y.C.) and INSERM (I.M.L.B., D.M., A.G. M, B.B, Y.C.), by the association Alsace contre le Cancer (I.M.L.B.). We thank the Fondation pour la Recherche Me´dicale [Grant number FRM FDT20170437233] and Eurostars for fellowships awarded to BB. Support and usage of platforms was provided by the French Infrastructure for Integrated Structural Biology [FRISBI, ANR-10-INSB-05-01], Instruct-ERIC, a Landmark ESFRI project (I.M.L.B., D.M., A.G.M, B.B, Y.C.) and by the French Proteomic Infrastructure [ProFI, ANR-10-INBS-08-03] (S.E, S.C.). Financial support was provided by GIS IBiSA and Re´gion Alsace in purchasing a Synapt G2 HDMS instrument (S.E and S.C.). The funders played no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors declare that they
particular contains the retinoid X receptor (RXR), the hepatocyte nuclear factor 4 (HNF4) and the COUP Transcription Factor 1 (COUP-TF)) and therefore that cannot be considered as monophyletic. This view separates the family into HNF4 on the one hand and all the other NRs on the other hand (Fig 1A). This phylogeny of early NRs now enables the study of the diversification and evolution of the various functions of NRs, such as ligand binding, DNA binding or dimerization. This was done for ligand binding and it allowed to propose that the ancestral NR was a sensor molecule capable of binding fatty acids with low affinity and low selectivity [9,12,13]. However, the same kind of evolutionary analysis has not yet been carried out to study the dimerization properties, a critical aspect of the nuclear receptors functions.
Thanks to their dimerization capability, NRs expanded the range of DNA target sequences through which they regulate target gene expression [1,2,14]. Several distinct dimerization properties have been characterized in NRs among which homodimerization on either palin-dromic or direct repeat DNA sequences, heterodimerization with RXR as a common partner, and even monomer binding (that is absence of dimerization) through the binding to extended half-site response elements (as depicted inFig 1B) [14]. The pivotal role of RXR (and of the insect homolog ultraspiracle protein (USP)) in this context has to be pointed out, as it is the promiscuous partner for more than 15 distinct high-affinity liganded NRs, including the reti-noic acid receptor (RAR), the thyroid hormone receptor (TR), the vitamin D receptor (VDR), the peroxisome-proliferator-activated receptor (PPAR), the liver X receptor (LXR) or the ecdy-sone receptor (EcR) in insects. Structural analysis revealed that NRs contain two separable dimerization interfaces, a relatively weak, albeit important interface in the DBD that plays a key role in DNA target site selection [15,16] and another stronger interface in the LBD. The detailed analysis of the LBDs dimerization interface highlighted the rules controlling homo-versus heterodimerization and allowed two functional NR classes to be defined according to their oligomeric behavior [17]. Class I NRs behave either as monomers or homodimers and exhibit a set of conserved residues that form a communication pathway linking helix 1 to the dimerization interface via helix 8. In contrast class II receptors encompass all NRs that hetero-dimerize with RXR and exhibit a different communication pathway linking the central helices H4/H5 to the dimerization interface via a conserved arginine residue in the loop between heli-ces H8 and H9 [17]. To deepen our understanding of how changes in NR dimerization proper-ties contributed to the diversification of the NR superfamily, we carried out an evolutionary analysis of NR genes, focusing on the evolution of dimerization across the entire NR superfam-ily. We show here that homodimeric binding was ancestral, whereas heterodimeric and mono-meric behaviors evolved later. We further identified a specific structural feature present in helix H7, a so-calledπ-turn or α/π-bulge, present in RXR and HNF4, as being an ancestral motif critical for the homodimerization of the most ancient NRs. We traced back the complex evolutionary history of thisπ-turn showing that it was instrumental in the origin of heterodi-merization, by allowing a flexible dimerization surface of RXR to accommodate numerous partners with multiple interfaces. Theπ-turn was originally used for homodimerization, but later was utilized for a different function, namely heterodimerization. This can be considered as a structural exaptation which can be seen as instrumental for the expansion of the repertoire of NR functions.
Results
A specific
π-turn motif is an ancestral feature of helix H7
suggesting strong structure-function constraints during evolution. A peculiar feature emerged from this structural analysis, which the presence of a helical deformation calledπ-turn or a α/ π-bulge within the α-helix 7 of RXR-USP and HNF4 LBDs (RXR, PDB: 1LBD, 6HN6 [18,19]; USP, PDB: 1G2N, [20]; HNF4), PDB: 1LV2, [21] (Fig 2).π-helices and π-turns account for over 15% of all known protein structures deposited in the PDB database [22–25]. Theπ-type helical structures are thermodynamically less stable thanα-helices and are considered to be favored only when they are associated with a functional advantage, typically for interactions with ligands or in the functioning of helical transmembrane domains. The occurrence of
π-Fig 1. (A) Simplified consensus tree of NR phylogeny based on Bridgham et al., 2010 [9]. (B) Some of the diversity found for the different DNA response elements (REs), illustrated here with a monomeric NR on an (extended) half-site; a homodimer on an inverted or an everted RE; and finally a heterodimer on direct repeat RE; N is the number of base pairs in the spacer between the two half-sites.
turns in the receptors that are considered to be at the origin of the NR family raises several questions, notably concerning the functional implications of this structural feature.
A conserved RxxxE motif, where the two invariant residues R and E form an intra-helical salt bridge further characterizes this specific conformation. In ahelical loop, also called a π-turn, the N+4 classical hydrogen bonds of theα-helix are replaced by N+5 hydrogen bonds [25,26]. Theπ-helical geometry results in the protrusion of the E residue out of the axis of the helix H7 with the two polar residues, E and R, closer to the helices H10-H11. Their side-chains form intricates inter- and intra-molecular interactions, stabilizing theper se energetically
unfa-vorableπ-helical conformation. The glutamate residue allows the formation of an intra-molec-ular salt-bridge with the conserved arginine residue of the motif. The arginine residue helps connect helix H7 to helices H10-H11 through binding to a conserved serine residue in helix H11 (S322 on the alignment, S427 in RXRαHS,S1 FigandTable 1). An additional hydrogen-bond is observed between theπ-turn and helices H10-H11. In RXR-USP, the H-bond is formed between E206 (E352 in hRXRα) and R316 in H10 (R421 in hRXRα) (Fig 2A). In HNF4, R202 (R267 in hHNF4α) binds to Q326 of H11 (Q350 in HNF4α) (Figs2BandS1and Table 1).
Theπ-bulge induced shift of residues only affects the N-terminal part of H7. The C-termi-nal side is anchored by a conserved bond between the carbonyl group of residue M/L214 (H7) and the side chain of R309 (H10). A similar type of interaction pattern prevails for both recep-tors and leads to strong interactions between H7 and H10-H11. These helices, together with the loop H8-H9 and helix H9, are the main contributors to the canonical NR LBD
Fig 2. The environment of theπ-turn in helix H7 of nuclear receptors. The environment of the π-turn is shown for (A) RXRα, (B)
HNF4α, (C) PNR and (D) COUP-TFII LBDs. The π-turn is shown in red, the π-turn motif residues (R202 and E206) are shown in orange. The 310helix at the H10-H11 junction in dark gray. In all cases, the C-terminal part of helix H7 is anchored to the N-terminal part of H10 by a conserved arginine (R309). Helix H7 is shown in blue and yellow ribbon representation for (A) RXRα and (B) HNF4α. The side chains of the signature motif residues, R202 and E206, and S322 of H11 form a triad of H-bonds (A) and (B). R316 at the H10-H11 junction in (A) RXRα and Q326 in H11 of (B) HNF4α complete the set of conserved bonds. For (C) PNR and (D) COUP-TFII, H11 is unstructured and the amino acid shift due to the absence ofπ-turn only affects the N-terminus of H7, while no major changes occur at the C-terminal part, in line with the structural alignment of the corresponding residues. In (C), PNR is depicted with H7 in light violet. Contacts between H7 and H10-H11 are only observed at the C-terminus of H7. In helix H11, a phenylalanine residue (F322) replaces the serine residue S322 of RXR that is important for interaction with theπ-turn. In (D), the original conformation of COUP-TFII is shown in green, the re-refined H7 conformers (seeMaterials and methods.) are shown in salmon and yellow for the straight and the curved helical conformations, respectively. A threonine residue (T322) in H11 replaces S322 of RXR H11 that interacts with theπ-turn. The figures are based on the PDB structures 1DKF for (A) RXRα, 4IQR for (B) HNF4α, 4LOG for (C) PNR and on the PDB structure 3CJW and our re-refined structure for (D) COUP-TFII.
https://doi.org/10.1371/journal.pgen.1009492.g002
Table 1. Amino acid residue mapping for nuclear receptors considered in this study. H stands for helices. ‘Alignment residue’ is the generic numbering used in this
study (S1 Fig). The class specific residues are specified by I and II for class I and class II NRs, respectively, with boxes colored in blue and green, respectively, or in cyan for residues common to class I and class II, together with the corresponding residue number [17]. The H7 column with yellow boxes refers to residues of theπ-turn, further highlighting the differential conservation of the p-turn.
Secondary structure H4-H5 H5 H7 H8 loop H8-H9 H10 H11 Class specific I (W) II (E,D) π-turn motif I; II (E) II (R) I (R) I; II (R)
Alignment residue 109 111 202 206 207 210 214 220 262 263 309 316 321 322 326
Brelivet numbering 40 42 - - - 50 61 62 93 100 105 106
-hRXRα W305 E307 R348 E352 L353 K356 M360 E366 D379 S380 R414 R421 R426 S427 K431
hHNF4α A224 E226 R267 E271 L272 P275 L279 E285 D298 A299 R333 L340 Q345 S346 Q350
hCOUP-TFII W249 E251 R293 E297 Q298 K301 L305 E311 D324 A325 R359 R366 R371 T372 S376
hPNR W257 E259 R301 E305 T306 R309 L313 E319 E332 T333 R367 L374 R379 F380 E384
hRARα C265 D267 D307 A311 F312 Q315 L319 E325 D338 R339 M373 K380 R385 S386 K390
hTRα C309 E311 D351 D355 L356 S359 F363 E369 D382 R383 F417 K424 R429 M430 C434
dimerization interface. Another interesting observation is worth mentioning: in RXR, the con-tact between the main chain of E206 (H7,π-turn) and the side chain of R316 (H10) occurs in a place where theα-helical conformation of H10 is locally changed to a short 3(10) helix charac-terized by N+3 hydrogen bonds. This peculiar 3(10) conformation of H10 is observed for all known NRs structures, except for the pregnane X receptor (PXR) and the steroidogenic factor 1 (SF1) that have classicalα-helices (e.g. PXR, PDB: 1ILG, [27]; SF1,PDB: 4QJR, [28]). In order to correlate the presence of the RxxxE motif with the occurrence of aπ-turn in H7, we carried out a structure-sequence analysis focused on H7 over several thousands of protein sequences. All available nuclear receptor sequences were taken into consideration. For 49 of them, at least one crystal structure was available. The RxxxE motif in H7 was found to be pres-ent in the NR2F group (COUP-TF, seven-up (SVP46/7-UP), V-erbA-related protein 2 (EAR-2)) as well as in the Photoreceptor-specific nuclear receptor (PNR) belonging to the subfamily NR2E (but not in FAX, and the tailless receptors (TLL or TLX)). Whereas no crystal structure is available for SVP and EAR-2 LBD, crystal structures were reported for COUP-TFII (PDB: 3CJW, [29]) and PNR (PDB: 4LOG, [30]). None of these structures exhibits aπ-turn confor-mation or a salt bridge between R and E residues of the RxxxE motif (Fig 2C and 2D).
In PNR LBD, H7 exhibits a canonicalα-helical conformation with no visible distortions. No intra-molecular interactions are seen between residues R and E of the motif. The serine res-idue observed in RXR H11 (S322) that is important for the stability of theπ-turn is replaced by F322 in PNR. This residue would generate a steric clash with aπ-turn conformer. If a π -helix would be present in PNR, the offset induced by the bulge would change the position of E200 that would then point into the direction of H5-H6, more specifically into a hydrophobic region composed of several leucine residues that would not favor interaction.
The
π-turn motif is ancestral and has been lost several times independently
The analysis of sponge nuclear receptor sequences show the presence of the RxxxE motif in helix H7 of SpNR1, but not of SpNR2. SpNR1 is associated with the group of nuclear receptors NR2B, C, D, E, F as well as NR3/4/5/6 subfamilies, while SpNR2 belongs to the HNF4-like sub-family. The markers of dimerization for class I and class II corroborate this interpretation (E5, W40, K/R55, R/K93, R105 for class I NRs; E/D42, R62, H/R/K90 for class II NRs and E50 and R105 universally conserved) [17]. Indeed, SpNR1 encompasses all of the class I markers, while in SpNR2, two class I markers (W40 and R105) are missing. Interestingly, the same class mark-ers are absent in HNF4. Homology modelling of SpNR1 using a reference panel of nuclear receptor structures, which in majority do not have aπ-turn, indicates the presence of a π-turn in 98% of the generated models (seeMaterials and methods). Furthermore, when SpNR1 replaces RXR in the structures of homodimers or heterodimers, the essential dimeric interac-tions are conserved. This suggests that the essential distinguishing features of RXR that can exist as a homodimer as well as a heterodimerization partner were already present in SpNR1.In order to understand the evolutionary dynamics of theπ-turn motif conservation, we plotted the presence of theπ-turn motif, as well as that of the RxxxE motif on a phylogenetic tree of NR sequences (Figs3AandS3). Our tree topology is fully consistent with previous stud-ies [8,9]. The tree allows to robustly position most NR subfamilies, even though a major unre-solved trichotomy still subsists concerning the branching of the NR3 and NR5/6 families relative to the robust NR7/NR4/NR1 cluster. Interestingly, our current sampling regarding sponges and other early metazoans sequences indicates that, within SpNR1, a lineage-specific amplification has occurred in calcareous sponges, leading to four distinct paralogues (num-bered P1 to P4 inS3 Fig). Our analysis therefore includes the whole currently known diversity of early NRs (seeS3 Fig).
Taken together, these data suggest that theπ-turn, and its associated RxxxE motif were present ancestrally in the primordial nuclear receptors and lost in several rapidly evolving line-ages of basal NRs (e.g. sponge SpNR2, some paralogous sponge SpNR1), as well as in the major
derived NR subfamilies (i.e. NR2EF, NR3, NR1 etc.).
The ancestral NR activated transcription as a homodimer
Nuclear receptors exhibit three different modes of oligomerization: homodimer binding (e. g.
steroid receptors or HNF4), heterodimers with the promiscuous partner RXR (e.g. TR, RAR, LXR or PPAR) and monomer binding (e.g. SF1 or Rev-erb) [1,31] (Fig 1B). It is important to note that these modes of binding are not mutually exclusive. For example, homodimer forma-tion has been demonstrated for Rev-erb which can also bind to DNA as a monomer [32]. Simi-larly, RXR can form either homodimers or heterodimers. This oligomeric behavior is related to the mode of binding to DNA, since response elements are derivatives of a canonical sequence (A/GGGTCA) that can be modified, extended or duplicated therefore offering a large palette of possible NR-selective binding modes [31]. As mentioned above, the final oligo-meric status is thus the result of the interplay between the strong dimerization interface in the LBD and a weaker one in the DBD which is crucial for response element selection [1]. To trace back the evolutionary history of the dimerization abilities of NRs, it is therefore necessary to fully disentangle the DNA binding and response element selection from the oligomeric status. For this reason, we focused our analysis on the major dimerization interface of the isolated LBD.
publications based on a similar dataset [8,9]. We adopted a conservative strategy in that when no experimental data was available, we coded the relevant oligomerization ability as unknown even if clear class I or class II residues can safely indicate the dimerization mode [17].
The ancestral state reconstruction for every node of the phylogeny illustrates successive complexification of the binding mode. Ancestrally, the binding mode is that of a homodimer, then only a heterodimer binding mode emerged once at the basis of the NR1 and NR4 families, while the monomer binding mode appeared several times independently from either from DR-homodimer or from RXR-heterodimers (Fig 3B).
The
π-turn residues are required for HNF4 biological function
According to the NR partition into class I (monomers and homodimers) and class II (heterodi-mers) NRs, RXR-USP and HNF4 belong to the class I. Class I differentially conserved residues (i.e. residues strictly conserved in class I and strictly absent in class II NRs) define a class-spe-cific interaction pattern that connects together H1 to H8 and H8 to H10, thereby networking the ligand binding pocket to the dimerization interface [17]. Examination of the class I-con-served residues in HNF4 indicates that this receptor is an outlier of the class I NRs. In fact, two class I invariant residues, W109 (W40 in the alignment given by [17]) and R321 (R105) are not conserved for HNF4, W109 being replaced by an alanine residue and R321 by a glutamine resi-due (Table 1). In HNF4, the residues A109 and Q321 are essentially conserved from cnidarians to mammals. In contrast, in sponge spNR2, W109 is replaced by a valine residue and R321 is mainly replaced by lysine or tyrosine residues but in a context lacking theπ-turn. Note that these two residues have important structural and functional roles. W109 is located at the
Fig 3. Evolution of the RxxxE motives compared to the dimerization modes during the history of the NR family. A. Evolution of RxxxE motives, as reconstructed
using ancestral mapping. B. Evolution of dimerization modes. Source files and script are provided in dryad under the following doi:10.5061/dryad.kkwh70s48.
junction of H4-H5, a highly conserved structural feature of the class I NR family and an inter-action hot spot for ligands. It was shown to be involved in a ligand-dependent allosteric mech-anism in RXR [37]. The arginine residue R321 in H10 is an important residue of the
dimerization interface highly conserved for all NRs, except in HNF4 and the oxosteroids sub-group (the androgen (AR), glucocorticoid (GR), mineralocorticoid (MR) and progesterone (PR) receptors). In the latter family, and only there, this mutation is associated with the muta-tion of the residue E111 (E42) that is normally strictly conserved in the whole NR family. In RXR, but not in HNF4, this amino acid residue binds to R321 and contributes to the stability of the homodimer. Altogether, the mutation of the two highly conserved residues W109 and R321 in HNF4 highlight the early divergence of this receptor from the rest of the family.
The analysis of the crystal structures of the HNF4α homodimer shows that a large contribu-tion to the stability of the homodimer comes from the unusual stacking of the tryptophan resi-due W325 (W349 in hHNF4α) in H10 with the corresponding resiresi-due of the other subunit (Fig 4A). These residues and the corresponding contacts they form are specific to this receptor family. Furthermore, several other residues of H10 belonging to one subunit contact helix H9 and the loop H8-H9 of the other subunit, thus forming a strong interaction network. In addi-tion, theπ-turn residue E206 in one subunit interacts with the region H10-H11, thereby form-ing intermolecular stackform-ing interactions with D262 (D298 in hHNF4α) in the loop H8-H9 of the other subunit (Fig 4A). In summary, we observe an intricate and unusual interaction net-work that involve residues of theπ-turn as well as helices H9 and H10 in both subunits. When we compared HNF4 to RXR, the numerous contacts (H-bonds, VdW. . .) that link together the two LBD subunits result in a larger buried surface at the dimer interface, consistent with an energetically more stable oligomer.
The functional importance of theπ-turn of HNF4 to the homodimerization process was demonstrated in earlier studies, where theπ-turn residues R202 and E206 were mutated and the functional consequence assessed [38]. In this work, it was shown that removing the charges of R202 and E206 impairs dimerization of the protein in solution and affect the HNF4α tran-scriptional activity in a variety of different cell lines. The impairment on trantran-scriptional activ-ity is even larger for the deletion mutantΔE206 (E262 in hHNF4α), which was also shown to be less efficient in recruiting transcriptional partners, such as SRC-1 and PGC-1. To correlate with biological effects, we searched the library of human HNF4 mutations reported for MODY1 syndrome and for various cancers that feature HNF4 somatic mutations. We found a small number of somatic mutations in theπ-turn motif, especially affecting R202 (R267 in hHNF4α), suggesting that this residue is indeed important for the biology of HNF4 in humans (HGMD database [39]).
Two specific features could explain absence of HNF4 heterodimers. First, the numerous H-bonds linking H9 and H10 of the LBD partners, mostly absent in RXR, are largely responsible for the strength of HNF4 homodimers, and much more stable than RXR ones (Fig 4A and 4B). Interestingly in RXR heterodimers, the number of bonds between helix H10 of RXR and helix H9 of the partner NR increases significantly (Fig 4C). Second, the mutation of a class I and II marker, R381 in RXR, Q321 in HNF4, is another remarkable feature. In RXR homo and het-erodimers, R321 is bound to serine 322, conserved in most class II partners.
The
π-turn motif is critical for RXR homodimer formation
other subunit. Importantly, theπ-turn residues play a crucial role in the stability of the homo-dimer. R202 and E206 are both involved in the dimerization interface. R202 link together S322 (H11) in the same subunit to R321 in H10 of the other subunit. E206 links together R316 (H10) found in the same subunit to D262 located in the loop L8-9 of the other subunit. The lat-ter residue further inlat-teracts with K210 located at the C-lat-ter of theπ-turn. Altogether, the
Fig 4. Schematic representation of the dimer stabilizing bonding interactions. The set ofα-helices H7 to H11 of each subunit of the LBD dimer is represented by cylinders. Water molecules are shown by red dots. Dimerization bonds between the two LBD subunits are represented by black lines and bonds between residues R202 and E206 of theπ-turn motif and residues at the dimer interface are shown by red lines. Shown in the scheme are bonds which are most frequently observed in the majority of the available structures. (A) HNF4α LBD. The dimer is symmetric and mainly stabilized by cross-contacts between helices H9 and H10. The π-turn contributes in an indirect manner to the dimerization interface through internal bonds with residues of helix H11. (B) RXR LBD. The dimerization is symmetrical, but less stabilizing bonds can be observed as compared to the HNF4 case. Interactions are also more uniformly distributed over the entire interface, but helix H9 is weakly involved in the dimerization interface, whereas theπ-turn is directly and strongly implicated. (C) RXR-RAR. The dimer is asymmetrical, and helices H9 are more strongly involved in the dimerization interface. The helix H7 of RXR directly contributes to the dimer interface through itsπ-turn. There is no contribution of RAR helix H7. (D.) USP-EcR. The interface is asymmetrical, but less asymmetric than for the different heterodimers formed with RXR. Note the unusual role played by helix H7 of EcR in the dimerization process [48].
structural analysis shows that theπ-turn residues are strongly involved in the homodimeriza-tion interface.
To assess the functional importance of theπ-turn residues for RXR homodimerization, we sought the effects of mutating the critical residues of theπ-turn motif on the dimerization behavior of RXR. To address this question, we chose to mutate E206 (E352 in hRXRα) of the RXXXE motif of RXRα LBD either into an alanine residue or to delete it completely from the LBD protein construct and relied on biophysical methods, including analytical size-exclusion chromatography (SEC), analytical ultracentrifugation (AUC) and native electrospray ioniza-tion mass spectrometry (ESI-MS) for the analysis of the oligomeric status of the wild-type and mutant proteins. In addition, we carried out molecular dynamics simulations of wild-type and mutant receptors to gain insights into the stability of the dimeric species.
The analytical SEC analysis was carried out using a S200 10/300 Superdex column by inject-ing the different proteins after the affinity purification step. The correspondinject-ing chromato-grams, shown inS4A Fig, reveal notable differences between wild-type (wt) RXRα LBD and E352A andΔE352 mutant constructs. The three proteins have a peak in common at an elution volume that roughly corresponds to the exclusion volume of the column (called void inS4A Fig), and therefore to large oligomeric protein species. Two additional peaks are observed for wtRXRα LBD (called peak1 wt and peak2 wt inS4A Figand indicated with red and blue sym-bols, respectively), whereas only one additional is seen for the mutants RXRα LBD peak (called peak mut inS4A Fig, and indicated with cyan and grey symbols for E352A RXRα and ΔE352 RXRα, respectively), with a similar elution volume. This indicates that the two mutant LBD constructs behave differently compared to wtRXR LBD and lack the larger species that com-pose peak 1 of wtRXR LBD. Since all SEC peaks correspond to pure protein samples, as shown in the SDS-PAGE gel in the insert ofS4A Fig, the difference in the size of the protein species composing each peak can solely be attributed to different protein oligomerization states and not to any co-purified contaminant species.
To further identify the SEC-separated species, SEC was online coupled to native ESI-MS for accurate oligomeric state assessment [40]. SEC-native MS analysis of wtRXRα LBD reveals two peaks, as shown inFig 5. The first peak (shown in red in the inset ofFig 5) consists of tet-ramers, whereas the second peak (shown in blue in the inset ofFig 5) is composed of dimers and monomers (Fig 5andS2 Table). In contrast, the main peak of both RXR mutants corre-sponds to dimeric and monomeric species only, while no tetramers are detected (Fig 5andS2 Table). Of note, under strictly identical experimental and instrumental conditions, theΔE352 RXRα mutant exhibits more dimers than the E352A RXRα mutant, which might suggest a slightly increased homodimer stability forΔE352. Altogether, the MS analysis indicates that mutating E352 of the RxxxE motif of RXRα LBD dramatically impairs noncovalent tetramer formation when compared to the wtRXRα LBD. However, we still observe a low abundance population of mutant RXRα LBD dimers in a large crowd of monomers. As strictly identical SEC columns could not be used off line and in-line with native MS, we further collected SEC peaks obtained on a S200 10/300 Superdex and analyzed the fractions by native ESI-MS (S5A– S5D FigandS3 Table). Again, native MS data analysis indicates that the wtRXRα LBD sample is composed of noncovalent tetramers and monomers (only low intensity dimers are detected) (S5A and S5B FigandS3 Table), whereas mutant RXRα LBD samples do not exhibit any tetra-mer species, but rather a mixture of monotetra-meric and ditetra-meric populations (seeS5C and S5D FigandS3 Table).
one peak for the other samples (shown in blue, cyan and grey inS4A Fig). Detailed examina-tion of the sedimentaexamina-tion data shows that for wtRXRα LBD, the SEC peak 1 is a heterogeneous sample with several species in dynamic equilibrium, including a majority of tetramers and smaller species down to the monomer, whereas the SEC peak 2 is composed of a mix of mono-mers and dimono-mers. In the case of the RXR mutants, the AUC data analysis indicates that the samples consist mostly of monomers and a slight amount of dimers. Thus, the two peaks observed in the differential sedimentation coefficient distribution c(S) correspond to the tetra-meric species for large S value and essentially to monomer species for the SEC peak 2 of wtRXRα LBD. For the mutants, monomeric species prevail, but the larger width of the c(S) peak suggests the formation of rapidly associating/dissociating dimers from the larger mono-mer pool. Importantly, no tetramono-mer is observed for the mutants RXRα LBD E352A and ΔE352. The AUC results show that monomers and tetramers are the main species of wtRXRα LBD, in full consistency with SEC-native MS observations (Figs5andS5A–S5D) native gel electropho-resis (S5E Fig). Note that a unique band is observed in the native gel of the mutant RXRα LBD
Fig 5. Oligomeric status of wild-type (wt) RXRα LBD and mutants, where the conserved Glu residue of the π-turn motif is mutated to Ala (E352A) or deleted (ΔE352). Size-exclusion chromatography (SEC)-coupled native mass spectrometric analysis for wt RXRα LBD
(red for the first peak and blue for the second peak), E352A RXRα LBD (cyan) and ΔE352 RXRα LBD (grey). The insert depicts the size-exclusion chromatograms of wt RXRα LBD and of mutants RXRα LBD (with the same color code). The region of the SEC peak considered for the integration of the mass spectra is shown as a line above the SEC peak. For wt RXRα LBD the tetrameric species is seen at the beginning (I) of the SEC chromatogram. In contrast, monomeric species is observed for all SEC peaks together with dimeric species. For mutant RXRα LBD species, no tetramer is observed in the SEC chromatogram and in the corresponding mass spectra where mainly monomeric and dimeric species are observed.
species which might be attributed to the rapidly exchanging monomer/dimer species or to the dominant dimeric fraction, as observed in the AUC and MS analyses.
Altogether, the biophysical data indicates that noncovalent tetramer formation is impaired for the RXRα LBD E352 mutants, in stark contrast with the wild-type protein. The RXRα tetra-mer is composed of a non-covalently bound ditetra-mer of RXRα homoditetra-mer and importantly rep-resents the main reservoir of RXRα homodimer available in the cell, as shown in vivo and in
vitro [41,42]. It was shown that disruption of the tetramerization interface of RXRα by mutat-ing conserved phenylalanine residues in helix H11 (depicted inS5F Fig) results in transcrip-tionally defective protein, without affecting the overall fold of the protein, nor ligand binding, dimer formation or DNA binding. Here, strikingly, we show that the mutation or the deletion of E352 in H7 impairs tetramer formation, by destabilizing the mutant RXRα homodimer spe-cies. This residue is far from the tetramerization interface composed of helices H3, H11 and H12 (S4F Fig) [43,44]. However, residues of theπ-turn interact with helix H11 and help stabi-lize its conformation. The mutation of the conserved Glu residue of theπ-turn does not pre-vent homodimer formation since the interface also encompasses other mostly conserved and hydrophobic residues [45]. However, it is likely to destabilize helix H11 and as a consequence to weaken the homodimer interface, enough to lead to the destabilization of the tetrameriza-tion interface, as observed experimentally.
We finally carried out Molecular Dynamics (MD) simulations to investigate whether the propensity for dimerization within RXR is affected. MD simulations of 50 ns were thus per-formed starting from a 1.9 A˚´ resolution crystal structure of HsRXR LBD (PDB: 1MVC [46]. Three sequences were used, including the E357A andΔE357 mutants. The total binding free energies were then calculated for each complex. The results, shown inS4 Table, suggest that wt RXRα LBD is the most stable homodimeric complex, followed byΔE352 RXRα LBD and
E352A RXRα LBD. Examination of the resulting structures after the MD simulations shows that for the E352 mutants, the contacts between H7 and H10 of one subunit and the loop H8-H9 and H9 of the other subunit are dramatically weakened (S6A–S6D Fig). Altogether, the biophysical characterization and MD simulations suggest that the E352 deletion and mutation has a destabilizing action of RXR homodimeric association, hampering tetramer formation for both mutants.
The
π-turn allowed RXR to evolve as a promiscuous partner for
heterodimerization
In contrast to HNF4α, RXRα can form heterodimers with NR partners. In all the cases, the heterodimerization interface is always asymmetric, whereby helix H7 of RXR is closer to the loop H8-H9 of the partner than the reverse. An intricate network of interactions spans the entire interface between RXR and its NR partner that involve helices H7, H8 and H9 and the loop H8-H9. The observation is consistent with experimental data indicating that the heterodi-mers are more stable than the RXRα homodimer [47]. The asymmetry of the dimerization interface has a direct impact on the number and the type of interactions. By taking RXRα/ RARα as an example (PDB: 1DKF [48]), we observe a scarce number of interactions between the loop H8-H9 of RXRα and helix H7 of RARα, while numerous interactions are seen in the reverse situation,i.e. between the loop H8-H9 of RARα and H7 of RXRα, and in particular its
π-turn in RXR and its concomitant absence in partner NRs. The asymmetry of the heterodi-mer together with the involvement of class conserved residues of the RXR partner in the het-erodimer interactions likely favored RXR as a common dimerization partner and led to the emergence of class II NRs as partners of RXR.
Finally, from the evolutionary point of view, it is interesting to consider the well-known ecdy-sone receptor, which is found in insects and other arthropods, in particular in insects and which is made of a heterodimer between EcR and USP, the ortholog of RXR. Several crystal structures of EcR/USP-RXR LBDs are available from different insect species [20,49–51]. All of the struc-tures exhibit an asymmetric heterodimeric interface, just like vertebrate NRs, with a similar interaction pattern as seen in the previous example of RARα/RXRα. In particular, helix H7 of USP-RXR that encompasses theπ-turn makes direct and water-mediated interaction with the loop H8-H9 of EcR (Figs4DandS7A). On the other hand, analyses of the structures indicate that, depending on the insect species considered, contacts between the helix H7 of EcR and USP-RXR may vary enormously [20,49–51]. In the more basal insect species, such as the beetle
Tribolium castaneum (Tc) (Coleoptera) and the silverleaf whitefly Bemisia tabaci (Bt)
(Hemi-ptera), no or few contacts are observed (none for Bt and one bond between H441 in H7 from EcR to Asp 325 in the loop H8-H9 of USP). In more recent species, such as the mothHeliothis virescens (Hv) (Lepidoptera), in stark contrast, numerous bonds are observed linking the helix
H7 of EcR and the loop H8-H9 and the helix H9 of USP-RXR (S7A Fig). The origin of this differ-ence between species comes from the position of the loop USP-RXR H8-H9 which is close enough for interaction with EcR in Hv, but not in Tc and Bt (S7B Fig). This discrepancy reflects more profound differences in the overall structure of USP-RXR among the different species [49,52]. In fact, USP-RXR of the basal insect species are more similar to the mammalian RXR than to sequences of USP-RXR of more recent species that encompass the Lepidoptera (moths and butterflies) and Diptera (flies, mosquitos) groups. Therefore, the peculiarity observed for HvEcR/HvUSP-RXR merely reflects the high evolutionary divergence of Lepidoptera and Dip-tera compared to the other clades [52–54]. However, the analysis of the more recent species EcR/ USP-RXR LBDs suggests that independently of the existing interactions made between H7 of EcR and USP-RXR, the asymmetry of the dimerization interface that is dependent of the pres-ence of theπ-turn in USP-RXR remains a conserved feature. Altogether, the analyses of the EcR/ USP-RXR structures nicely illustrate the evolutionary conservation of the heterodimerization interface, its asymmetry and the involvement of theπ-turn into the dimerization mechanism.
To substantiate our hypothesis for the role played by theπ-turn in the heterodimerization, we experimentally characterized the heterodimers between RXRα LBD and PPARα LBD for the wild-type and the E352 RXRα mutants. We used SEC-coupled to native MS to relatively quantify the heterodimeric PPARα/RXRα population in the complex mixture between RXRα (wt or mutant) and its partner PPARα LBD.Fig 6summarizes the relative abundances of monomeric and heterodimeric species as deduced from native MS results (S8 Fig). All RXRα construct (wt but more interestingly also E352 mutants) allow formation of PPARα/RXRα het-erodimers (Figs6and S8, together with the presence of monomeric RXRα and PPARα. How-ever, there is a strong reduction in the relative PPARα/RXRα heterodimer population between PPARα/wtRXRα and PPARα/E352 mutants (Figs6andS8), along with increased amounts of free monomeric RXRα detected.
Discussion
The
π-turn clusters a crucial interaction network in basal NRs
network of amino acid residue interactions, its complex evolutionary history and conservation in basal NRs strongly suggest that it is a key structural element for the functional diversifica-tion of NRs. Our structural analysis reveals that theπ-turn, located within helix H7, is always associated with the presence of an RxxxE motif. Furthermore, our 3D homology modeling study allowed us to infer the presence of aπ-turn in NRs that harbor the RxxxE sequence motif, but for which no structural and functional data are available. As a result, we observed the presence of aπ-turn in HNF4 of bilaterians and basal metazoans, such as cnidarians and placozoans (Trichoplax), as well as in RXRs of bilaterians and cnidarians and in SpNR1. The
latter case is particularly interesting, since SpNR1 together with SpNR2 represent the only NRs found in sponges. These two receptors, which are used to root the NR superfamily tree, are considered to be the most basal NRs and thus define the two major subdivisions in the NR evo-lutionary tree, one containing SpNR2 and HNF4 (NR2A) and the other one containing SpNR1 and all the other NRs (Fig 3A) [9].
Our phylogenetic analysis enabled us to hypothesize that theπ-turn is an ancestral motif that was present early in the primordial NRs and that was further differentially lost at least five times during the NR evolution. Due to the similarities between theπ-turn present in the struc-tures of RXR and those of HNF4, we strongly support the “π-turn early” scenario, rather than the alternative “π-turn late” scenario of the late independent origin of the π-turn in HNF4s, RXRs and SpNR1 (Fig 3A). Based on our analysis, we inferred that aπ-turn similar to those seen in HNF4 and RXR should be present in SpNR1. A crystallographic study of SpNR1 LBD would be the ideal test for our hypothesis.
The structure-sequence analysis of NRs that exhibit a RxxxE motif indicates that among all NRs whose structure is known, only RXR-USP and HNF4 possess a peculiarπ-helical
Fig 6. Heterodimerization capacity of wild-type (wt) RXRα LBD and mutant E352A and ΔE352 RXR LBD with PPARalpha. Size-exclusion chromatography
(SEC)-coupled native mass spectrometry (MS) analysis for complex mixture of PPARα LBD with either (A) wt RXRα LBD, (B) E352A RXRα LBD or (C) ΔE352 RXRα LBD. The isolated RXRα LBD (wt or mutants) is depicted in yellow, isolated PPARα LBD in blue and heterodimeric PPARα/RXRα LBDs in green. Mass spectra obtained by deconvolution of the raw data (shown inS6 Fig) for the three different complex mixtures are shown, together with the quantification of the species from the SEC-native MS analysis of the PPARα/RXRα LBD complex mixture, shown below in the form of histograms.
geometry. This intrinsically unstableπ-helical conformation requires strong stabilizing inter-actions between the N- and the C-terminal parts of H7 and neighboring regions in its molecu-lar environment to hold together this topological feature. Importantly, theπ-turn of H7 thus gives rise to specific intricate interactions with helices H10 and H11, both being crucial ele-ment of the dimerization interface. As a matter of fact, the junction between the two helices H10-H11 encompasses a 3(10) conformation and a specific leucine rich sequence (LLLXXL or LLXXL) at the N-terminal part of H10. These structural features, which induce a kink in the region of helices H10-H11, make possible crucial and complementary interactions between theπ-turn conformer of H7 and H11, notably between the arginine residue of the RxxxE motif and the serine residue of H11. Therefore, theπ-turn is at the heart of the network of interac-tions present in RXR and HNF4 from the origin for the stabilization of the LBD and for the formation of a stable homodimerization interface (Fig 4). These interactions allowed the ancestral receptor to bind DNA response elements as a dimer in a cooperative manner, an abil-ity that increased the DNA binding site selectivabil-ity. It is important however to emphasize that theπ-turn is not necessary for the homodimerization of all nuclear receptors, but only for RXR and HNF4. In fact, steroid NRs, such as the estrogen receptor (ER) and the estrogen-related receptor (ERR) homodimerize in the absence ofπ-turn and RxxxE motif, making use of the same secondary structural elements for building the dimerization interface as RXR and HNF4. From an evolutionary point of view, ER and ERR evolved in a way such as they under-went compensatory mutations leading to the disappearance of the RxxxE motif, but conserving most of the other interacting residues. On the other hand, the later evolved oxosteroid nuclear receptors (AR, GR, MR and PR) are different in their dimerization properties. Their ligand binding domain does not dimerize in the same manner as ER and ERR. In fact, there is a marked sequence difference of the residues at the interface compared to the whole nuclear receptor family and the presence of an additional conserved region at the C-terminal end of the LBD that hampers the oxosteroid receptors to dimerize in a classical way [1,55] that still needs to be uncovered.
In protein structures,π-helices and π-bulges are often associated with a specific function, making them powerful markers of protein evolution [23]. An accepted hypothesis about the emergence ofπ-bulges is their frequent implication as ligand binding site contributors such as in GPCRs (van der Kant and Vrient, 2014). In the case of NRs, a direct association with ligand binding is rather unlikely. For example, both apo and holo crystal structures are available for RXR and, importantly, show no significant differences in theπ-turn environment. Note that thein vivo relevance of RXR ligands, such as 9-cis retinoic acid or DHA, is a highly debated
and controversial issue [56]. Similarly, whereas all known HNF4 crystal structures are liganded, the biological significance of HNF4 ligands is not clear, since the latter are either non-exchangeable molecules found in the LBD structure or do not induce any transcriptional activity [57]. For SpNR1 and SpNR2, barely no information is available. Functional characteri-zation of sponge receptors combined with phylogeny analysis and ancestral sequence recon-struction allowed Bridghamet al. to propose that NRs evolved from a ligand-activated
ancestral receptor that existed near the base of the Metazoa, with fatty acids as possible ances-tral ligands [9]. Taken together, these data indicate that the presence of theπ-turn in RXR and HNF4 is not related to the ligand binding capability.
many other NRs. For all of them, the adaptation of the pocket to the ligand occurs through substantial changes of the region encompassing helices H7 and H11, and theβ-sheet. Focusing on helix H7, the structural adaptation of this helix can occur only when it is devoid of the structural constraints that would be imposed by the presence of aπ-turn. In other words, molding and adaptability to various ligand molecules is correlated to the absence of aπ-turn. Thus, from the evolutionary point of view, the disappearance of theπ-turn in more recent nuclear receptors (from NR1 and NR4 subfamilies) facilitated the binding of a variety of mole-cules and promoted their diversification.
The presence of aπ-turn in RXR and HNF4 suggests that its maintenance is linked to a dif-ferent function, namely dimerization (Fig 7). Our analysis supports the key role of theπ-turn of RXR in heterodimer formation through numerous interactions with the loop H8-H9 and the helices H9 and H10 of the partner NR. Experimental evidence provided here for the case of PPARα/RXRα LBD fully supports our hypothesis. The lack of π-turn in the partner receptor strengthens the resulting asymmetric heterodimer. We hypothesize that the presence of the π-turn in RXR is a necessary condition for this receptor to be the ubiquitous dimerization part-ner of many different NRs. This structural feature is namely linked to a stiffening of the LBD structure, especially the heterodimerization region, allowing RXR to dimerize in a similar fash-ion with different partner receptors.
The
π-turn represents an unusual exaptation
In protein science, it has always been thought that theπ-turn is a structural feature that evolved in a way such as to accommodate novel functionalities. This is not the case here, since the π-turn is an ancestral motif that was instead lost during NR diversification. However, its presence or its absence is linked to critical biological functions. On the one hand, the presence of the π-turn in the most ancestral receptors is crucial for the stabilization of a homodimer interface in the context of small molecule binding in the LBD for sensor function. On the other hand, the loss of theπ-turn in all subsequent NRs allowed their binding site to adapt to a different type of ligands and for a large group of them facilitated their heterodimerization with RXR in a stronger and asymmetric manner.
which a dimerization motif evolved from a biotin binding domain [71]. Here, we propose that theπ-turn, an ancestral structural feature that was present in ancient receptors, in particular in RXR and HNF4, was important for homodimerization and later utilized by RXR as a key struc-tural element for heterodimerization. However, its loss allowed the reinforcement of ligand adaptation in other NRs. Both contrasting aspects eventually led to a substantial expansion of the repertoire of NR regulatory abilities.
To summarize, both the presence (in RXR) and the absence (in NR partners) of theπ-turn led to the emergence of new NR function, namely the heterodimerization of RXR with partner receptors, leading to greater target site selection and the emergence of high affinity receptors due to more flexible binding site that could diversify in terms of ligand binding possibilities. We propose that theπ-turn in NRs represents a case of structural exaptation, namely a trait whose benefit for the system is unrelated to the reason of its origination, but which allowed an unprecedented increase of the NR regulatory repertoire.
Materials and methods
Structure refinement of COUP-TF LBD
A careful analysis of the crystal structure of COUP-TFII and its corresponding electron density map reveald that large portions of the electron density in the region of H7 could not be inter-preted. The main problems are located between H5 and H7, with no visible electronic density for theβ-sheet and H6 connecting H5 to a disordered N-terminal part of H7. For the latter a closer inspection to the electron density map suggests that the N-terminal part of H7 could adopt different conformations. Since this region was critical for our analysis of the RxxxE motif and the structural features associated to it, we decided to further improve the protein structure around this location by iterative building in Coot of residues in the non-interpreted electron density map followed by a crystallographic refinement using Phenix. This work resulted in better crystallographic quality factors R and Rfree and to a more confident interpre-tation of the electron density map (seeS1 Table).
After crystallographic re-refinement, we observe that in the crystal packing helix H7 can adopt two helical structures, together with a lengthening of helix H7 at its N-terminal side as compared to the original helix of the PDB structure (S2 Fig). The two novel conformations correspond to a regular straight and a curvedα-helix bent at the level of the putative π -turn. The C-terminal parts of the two helices overlap nicely, while their N-terminal ends are located over 6Å apart. These conformations are in equilibrium in the crystal, alternating between nearest neighbour molecules to ensure optimal packing and are likely to be natural conforma-tions. The dynamics of H7 resulting from the absence of a stabilizing H11 promotes the adapt-ability to packing constraints with a subsequent disorder of this subdomain. In fact, the lengthening of the original single helix H7 to the size of the re-refined one would lead to steric clashes between crystallographic dimers.
Fig 7. Evolution of theturn motif and dimerization interface. (A) Evolutionary history of the main receptor families in regard to
π-turn and dimerization. The rectangles correspond to LBD monomers and their color indicates their class marker composition: red (e.g. RXR) for all Class I markers, green (e.g. HNF4) when the W109 and R321 are missing, and blue (e.g. NR7) for other Class I marker anomalies. The first NR is indicated in black as no information is available. The blue numbered circles are duplication events that are plotted on the tree on panel B. The ligand binding pocket is indicated as a green circle when it is liganded and a blue cross when there is no or weak non-specific interaction. Theπ-turn motif is indicated by a big PI symbol (π), red for motif and structure present, black for motif present but no information about structure, blue for motif present and structure absent and ‘?’ symbol when no information is available. (B) Phylogenetic position of the evolutionary events described in (A) (blue numbered circles). For porifera, placozoans and cnidarians, the minimal sets of receptors present in their last common ancestor are indicated.
The second important observation is the absence of theπ–turn. Although the electron den-sity was not clear enough to confidently build the side chain of R293, some denden-sity can be seen that could correspond to the guanidinium group of the arginine in the straight conformation of the helix, indicating that in this conformation, the intra-helical salt bridge between the side chains of the arginine and the glutamic acid of the RxxxE motif could be maintained. How-ever, this intra-helical salt bridge is rotated to a position such that no interaction between the H7 motif and H10-H11 can take place. The shift induced by the absence of theπ -turn prevents E206 from binding R316, instead the connection is made with its neighboring residue Q207 (Q298 in hCOUP-TFII). The conserved serine residue of RXR H11 that stabilizes theπ -turn in RXR-USP and HNF4 is replaced by a threonine residue, but without interacting with H7 residues. Furthermore, no interactions are seen between H7 and H5-H6. Of note, helix H7 after refinement does not exhibit a 3(10) helical turns as suggested in the original structure.
Evolutionary analysis
Collected NR sequences were aligned using Clustal Omega (Sievers and Higgins, 2014) and alignments were checked manually and edited with Seaview (Gouy et al., 2010). Phylogenetic trees were built using PHYML (Guindon and Gascuel, 2003). Following model testing using AIC and BIC criteria as implemented in the SMS software (Lefort at al., 2017), we selected the LG model (Le and Gascuel, 2010) with a gamma law and estimation of the proportion of invariable sites. The reliability of nodes was assessed by likelihood-ratio test (Anisimova and Gascuel, 2006). Ancestral character reconstruction and stochastic mapping (Huelsenbeck et al., 2003) were performed under R version 3.2.2 (R Core Team, 2015) using the make.sim-map function as implemented in the phytools package version 0.5.0. (Revell, 2012). Character evolution was inferred using a model of symmetrical transition rates between the character states (SYM). 10 000 character histories were sampled to allow the incorporation of the uncer-tainty associated with the transition between different states. Inferred state frequencies for ancestral nodes were plotted using the describe.simmap function. Commands and sources files for ancestral mapping are provided in the Dryad repository under the following doi:10. 5061/dryad.kkwh70s48.
Cloning, expression and purification for biophysical studies
HsRXRα LBD, wild type (T223-T468) and mutants E352A and ΔE352, were cloned into the pET15b expression vector. HsPPARα LBD (I195-Y468) was cloned in a pET15b expression vector. Each individual vector was transformed intoEscherichia coli BL21 (DE3), grown at
37˚C and induced for protein expression at an OD600nmof 0.6 with 1 mM IPTG at 25˚C for 3 hours. The corresponding cell pellet was resuspended in binding buffer (20 mM Tris pH = 8.0, 400 mM NaCl, 10% glycerol, 2 mM CHAPS, 5 mM imidazole) and lysed by sonication. The crude extract was centrifuged at 45’000 g for 1 hour at 4˚C. The lysate was loaded on a Ni affin-ity step on HisTrap FF crude column (GE Healthcare, Inc.) and the protein was eluted at a concentration of 150 mM imidazole. The LBD protein was then polished by size-exclusion chromatography in a SEC buffer (20 mM Tris pH = 8.0, 250 mM NaCl, 2 mM TCEP) by using a Superdex S75 16/60 column (GE Healthcare).
Polyacrylamide native gel electrophoresis
with its DNA counterpart at defined molar ratios.The polyacrylamide gels were stained using Instant Blue Protein Stain (Expedeon Protein Solutions) for 15 min and rinsed in water.
Analytical ultracentrifugation
Sedimentation velocity experiments were conducted using Beckman Coulter ProteomeLab XL-I analytical ultracentrifuge using the 8-hole Beckman An-50Ti rotor at 4˚C for samples in a buffer composed of 20 mM Tris pH = 8.0, 250 mM NaCl, 20μM TCEP [72]. The molar pro-tein concentration of the experiments corresponds to 1μM. Sedimentation at 50000 rpm was monitored by absorbance at 220 nm with scans made at 5 min intervals. The solution density and viscosity for resuspension buffer were calculated using SEDNTERP software. Data were analyzed using a c(s) model in SEDFIT [73].
Size-exclusion chromatography hyphenated to non-denaturing mass
spectrometry (SEC-non denaturing MS)
For SEC-non-denaturing MS analysis, an ACQUITY UPLC H-class system (Waters, Manches-ter, UK) comprising a quaternary solvent manager, a sample manager cooled at 10˚C, a col-umn oven maintained at room temperature and a TUV detector operating at 280 nm and 214 nm was coupled to the Synapt G2 HDMS mass spectrometer (Waters, Manchester, UK). 50μg of each samples were loaded on the ACQUITY UPLC Protein BEH SEC column (4.6× 150 mm, 1.7μm particle size, 200 Å pore size from Waters, Manchester, UK) using an isocratic elu-tion of 150 mM ammonium acetate (NH4OAc) at pH 7.4 and at a flow rate of 0.25 mL/min over 4.0 min. Then the flow rate was decreased to 0.10 mL/min over 5.9 min and finally increased to 0.25 mL/min over 1.9 min. The Synapt G2 HDMS was operated in positive mode with a capillary voltage of 3.0 kV while sample cone and pressure in the interface region were set to 40 V and 6 mbar, respectively Acquisitions were performed in 1,000–10,000 m/z range with a 1.5 s scan time. The mass spectrometer was calibrated using singly charged ions pro-duced by a 2 g/L solution of cesium iodide (Acros organics, Thermo Fisher Scientific, Wal-tham, MA USA) in 2-propanol/water (50/50 v/v). Native MS data interpretations were performed using Mass Lynx V4.1 (Waters, Manchester, UK). For PPARα/RXRα experiments, the two nuclear receptors were expressed in E. coli and purified separately. Mixtures of 1:1 molar ratio of RXRα (wt or mutants) and PPARα LBD were performed in the purification buffer, incubated and injected on the SEC column coupled to the mass spectrometry instru-ment as described above. Deconvolution was performed using UniDEc [74]. Relative abun-dances of the species were calculated from native MS intensities of the deconvoluted data.
Off-line native electrospray-mass spectrometry (ESI-MS)
v). Native MS data interpretations were performed using Mass Lynx V4.1 (Waters, Manches-ter, UK).
Molecular modeling of sponge NR1
Homology modeling of the NR1 sequence fromAmphimedon queenslandica (ACA04755.1)
using multiple templates was performed using Modeler (Webb and Sali, 2016) in order to eval-uate the possible three-dimensional fold of this sequence, in particular whether aπ-turn could be formed. The multiple alignment was constructed using 15 PDB structures of RXR (1MV9, 1H9U), USP (1Z5X, 1G2N, 1HG4), HNF4 (1LV2, 1PZL), LRH (1PK5, 1YUC), RAR (1DKF, 1FCY, 1XAP), TR (1NAV), ERR (1S9P) and LXR (1UPV). Sequence identity between NR1 sequence and these templates ranged from 27% (ERR) to 42% (RXR). A total of one hundred models were generated and evaluated according to their DOPE scores. The model with the best score, as well as all other models obtained contain aπ-turn in helix H7, with very similar orientations of R and E residues when compared to RXR (obtained in the structure PDB code 1DKF). Since sequence identity is very elevated between NR1 and RXR and HNF4 sequences, another homology model was built using the same multiple alignment with only sequences of receptors without aπ-turn and consisted of LRH (1PK5, 1YUC), RAR (1DKF, 1FCY, 1XAP), TR (1NAV), ERR (1S9P) and LXR (1UPV). Similarly, a total of one hundred models were cal-culated and their quality evaluated according to their DOPE scores. In this case, the best model does not contain aπ-turn in H7, however when comparing the scores of both best mod-els from the two modeling strategies, the model withπ-turn has the best score (-27875.7) with respect to the one without aπ-turn (-27771).
Model assessment was also calculated using ProQ2 (Ray et al., 2012), an algorithm predict-ing local and global quality of protein models, based on properties from sequence (predicted secondary structure for example) and structure (atom-atom contacts, residue-residue contacts, secondary structure). This algorithm provides a score for each residue and was used to assess the quality of the homology modeling specifically for helix H7. We calculated the average score of helix H7 (ranging from 0 to 1, the latter being the best score) and obtained 0.71 and 0.65 with standard deviations of 0.06 and 0.04 for the models with and withoutπ-turn respectively, supporting an enhancement in quality with the presence of theπ-turn in the nuclear receptor structure.
Homology modeling using multiple templates was performed using Modeler (Webb and Sali, 2016), sequences with an e-value of 0 (best alignment) were extracted from non-redun-dant PDB sequences. The multiple alignment was constructed using 15 PDB structures of RXR (1MV9, 1H9U), USP (1Z5X, 1G2N, 1HG4), HNF4 (1LV2, 1PZL), LRH (1PK5, 1YUC), RAR (1DKF, 1FCY, 1XAP), TR (1NAV), ERR (1S9P) and LXR (1UPV). Sequence identity between NR1 sequence and these templates ranged from 27% (ERR) to 42% (RXR). A total of one hun-dred models were generated and evaluated according to their DOPE scores. Another homol-ogy modeling run was performed using structures without aπ-turn in helix H7 and consisted of LRH (1PK5, 1YUC), RAR (1DKF, 1FCY, 1XAP), TR (1NAV), ERR (1S9P) and LXR (1UPV). Similarly, a total of one hundred models were calculated and their quality evaluated according to their DOPE scores.
Molecular Dynamics simulations
for theΔE352 sequence. A model without a π-turn was thus generated, and we insured that all other side chains aside from this region remained similar to the wild-type structure. Hydrogen atom placement was performed using the HBUILD facility [75] in the CHARMM program [76]. All three structures were solvated in cubic boxes of approximately 121 per side, with a salt concentration Na+/Cl- corresponding to the physiological concentration of 150mM. Before solvating the system, two minimizations of 100 steps of Steepest Descent method and 1000 steps of Adapted Basis Newton-Raphson method were performed in order to eliminate steric clashes.
Molecular dynamics simulations were performed using the CHARMM36 force field [77] within the NAMD program [78], following two steps. First, minimization and heating of water molecules around the fixed protein was realized with 1000 steps of Conjugate Gradient (CG) energy minimization, heating up to 600K over 23ps, 250 steps of CG energy minimization, and heating to 300K over 25ps. Second, positional restraints on the protein were removed and all the system was energy minimized with 2000 steps of CG and heating to 300K over 15ps, fol-lowed by 85ps of equilibration. The production run was then performed for the duration of 50ns. Periodic boundary conditions were used and the particle mesh Ewald algorithm [79] was applied to take into account long-range electrostatic interactions. All bonds between heavy atoms and hydrogens were constrained using the SHAKE algorithm [80] and an integration time step of 2fs was used for all simulations. This protocol has been carefully benchmarked across different nuclear receptor proteins, such as RAR, ER, GR [81–85]. The first 10ns of the production run were excluded from all analysis to ensure proper equilibration. Time evolution of Cα-RMSD of the three systems wt RXRα LBD, ΔE352 RXRα LBD, and E352A RXRα LBD across the three simulations are represented inS6D Figto illustrate the stability of the struc-tures over the course of the analyzed timeframe.
Binding free energies were estimated on the average structures, calculated over the time frame between 10 to 50ns. We used the University of Houston Brownian Dynamics (UHBD) [86], to solve the linearized Poisson-Boltzmann equation and compute the electrostatic bind-ing free energy of bindbind-ing of the two molecules. A dielectric constant of 80 was used for the solvent, 1 for the protein and a pH of 7. Van der Waals radii and charges of atoms are obtained from the force field CHARMM36. A nonbonded cutoff of 12.5Å were used with a shift trunca-tion functrunca-tion for electrostatics. Although MM/PBSA does not take into account conforma-tional entropy, our protocol [87] has been validated on a variety of systems to assess protein/ protein complexes in terms of relative free energies and proven to be in agreement with experi-mental data [84,85].
Supporting information
S1 Fig. Reference sequence alignment snippet used in this article. Alignment generated with
Clustal Omega software and manually corrected. (TIF)
S2 Fig. Helix H7 of the ligand-binding domain of COUP-TFII displays a double conforma-tion. (A) Ribbon diagram showing the double conformation of helix H7. Conformation A is
colored in green, and conformation B in blue in all images. All images are presented in cross-eye stereo. (B) 2mFo-DFc electron density map contoured at 0.5 sigma (0.16 e-/Å3) around helix H7. The density for conformation B is shown in blue, and the supplemental density for conformation A is shown in green. (C) 2mFo-DFc electron density map contoured at 0.5 sigma (0.16 e-/Å3) around conformation B only of helix H7. (D) 2mFo-DFc electron density map contoured at 0.5 sigma (0.16 e-/Å3) around conformation A only of helix H7.
